The long-term goal of this project is to characterize the mechanisms that specify the fate of the germline, a fundamental problem in developmental biology. In all animals, the founder cells of the germline (primordial germ cells) display two conserved characteristics: they possess germ granules, RNA granules specific to the germline, and they express members of the Nanos family of RNA-binding proteins. The goal of this proposal is to understand how germ granules and Nanos function together to specify the fate of primordial germ cells. In preliminary work, we have found that PGCs that lack Nanos activity prematurely activates the expression of ~1000 genes normally expressed in oocytes and somatic cells, a phenotype also seen in mutants that lack the Polycomb Repressive Complex. Specific Aim I will identify the mechanisms used by Nanos to prevent this massive gene mis-regulation. We have already identified one promising candidate Nanos target: LIN-15B, a likely component of the DRM transcription factor complex that activates gene expression in oocytes. Specific Aim II will examine the role of a new group of germ granule proteins we recently characterized. The MEG proteins form stabilizing scaffolds around the central cores of each germ granule. Our genetic analyses indicate that the MEGs, but not the proteins in the core, are essential for PGC fate. We will investigate how the MEGs, predicted to be intrinsically-disordered with no recognizable domain, function with Nanos to specify PGCs. Two technical breakthroughs support this application. First we have worked out methods to isolate PGCs in large enough numbers to determine their transcriptome by RNAseq. We will use this method to define how Nanos and germ granule proteins affect the PGC transcriptome at a genome-wide level. Second we have developed a highly efficient genome editing method, using CRISPR/Cas9, that allows us to mutate, tag, delete and replace any gene of interest in just 4 days. This method gives us unprecedented genetic and biochemical access to the genes and proteins that are the focus of this application.